Determining the role of CLN3 in the eye

确定 CLN3 在眼睛中的作用

• 批准号: 10357934
• 负责人: Ruchira Singh
• 金额: $ 45.82万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10357934
• 关键词: Affect; Animal Model; Apical; Atrophic; Binding; Blindness; CLN3 gene; Cadaver; Cell Death; Cell Fractionation; Cell physiology; Cell surface; Cells; Cellular Structures; Chronic; Clinical; Data; Dextrans; Digestion; Disease; Epithelial; Epithelial Physiology; Evolution; Excision; Eye; Family suidae; Functional disorder; Genotype; Health; Homeostasis; Human; Image; Immunohistochemistry; Impairment; Ingestion; Knowledge; Laboratories; Lead; Light; Link; Lipofuscin; Longitudinal Studies; Mediating; Molecular; Mus; Mutation; Nerve Degeneration; Nutrient; Pathology; Patient imaging; Patients; Pattern; Phagocytosis; Pharmacotherapy; Phenotype; Photoreceptors; Play; Proteins; Recycling; Retina; Retinal Degeneration; Retinal Photoreceptors; Rodent; Role; Secondary to; Structure; Structure of retinal pigment epithelium; Techniques; Testing; Transportation; Vision; Vitamin A; Waste Products; cellular imaging; cellular microvillus; disease-causing mutation; early childhood; experimental study; feeding; gene correction; histological studies; imaging study; in vivo; induced pluripotent stem cell; insight; internal control; juvenile neuronal ceroid lipofuscinosis; mouse model; multimodality; novel; porcine model; retinal imaging; retinal progenitor cell; uptake; visual cycle

ABSTRACT Juvenile neuronal ceroid lipofuscinosis (JNCL), caused by mutations in the CLN3 gene, leads to neurodegeneration and vision loss in early childhood. Vision loss in JNCL is due to retinal degeneration. Specifically, in the retina, JNCL affects both the light sensing photoreceptor cells and their underlying epithelium, RPE. Furthermore, photoreceptor cell loss in the JNCL retina has been linked to vision loss. However, the molecular mechanism(s) underlying photoreceptor cell loss in JNCL are currently not known. Preliminary studies in our laboratory utilizing human induced pluripotent stem cell (hiPSC)-derived retinal cells suggest a novel role of CLN3 in maintaining key RPE structure (microvilli) and function (uptake of shed photoreceptor outer segments or POS) that are essential for photoreceptor survival and therefore vision. Specifically, JNCL hiPSC-RPE display microvilli disorganization/ballooning and impaired phagocytosis of POS compared to control hiPSC-RPE cells. Notably, consistent with RPE microvilli dysfunction and reduced POS uptake in JNCL hiPSC-RPE cells, RPE in the eyes of JNCL patients have reduced lipofuscin (breakdown products of POS digestion). Together, these data suggest that “CLN3 is a RPE microvilli resident protein that regulates POS uptake and CLN3 dysfunction in JNCL leads to impaired POS phagocytosis and subsequently decreased lipofuscin accumulation in JNCL RPE cells.” To test this hypothesis, in this project, we propose to employ ultrastructural and molecular studies on control and JNCL hiPSC-RPE cells. Specifically, we will first utilize immunohistochemistry, RNAScope and subcellular fractionation techniques to confirm the localization of a proportion of CLN3 in RPE cells to the apical microvilli in primary (mouse, porcine, human) and control hiPSC- RPE. Next, we will compare i) microvilli structure and molecular composition and ii) POS binding and internalization by control vs. JNCL hiPSC-RPE cells. Furthermore, to correlate the reduced POS phagocytosis by JNCL hiPSC-RPE cells to reduced lipofuscin (autofluorescence accumulation) seen in JNCL RPE in vivo, we will evaluate the pattern of autofluorescence accumulation (cell surface vs. intracellular) in parallel cultures of control and JNCL hiPSC-RPE after chronic POS feeding (1-3 months). We will also validate our findings on patient hiPSC-RPE cells using primary RPE/POS from a JNCL mouse and porcine model. Finally, we will utilize in vivo retinal imaging of patients with JNCL in a longitudinal study to document the presence and amount of autofluorescence/lipofuscin in the photoreceptor-RPE cell layer in relationship to photoreceptor vs. RPE cell loss in patients progressing through the disease. Altogether, the knowledge gained from this study will provide novel insights into i) the role of CLN3 in RPE physiology and ii) the plausible contribution of RPE dysfunction to JNCL retinal pathophysiology. !

抽象的 由CLN3基因突变引起的少年神经元脂肪促脂肪促（JNCL）导致 儿童早期的神经变性和视力丧失。 JNCL的视力丧失是由于永久性变性。 具体而言，在视网膜中，JNCL影响光感应感光细胞及其基础 上皮，RPE。此外，JNCL视网膜中的感光细胞损失与视力丧失有关。 然而，目前尚不清楚JNCL中基础感光细胞损失的分子机制。 使用人类诱导多能干细胞（HIPSC）衍生的残留细胞的实验室初步研究 提出CLN3在维持关键RPE结构（微绒毛）和功能（棚的吸收）中的新作用 光感受器的外部段或POS）对于感光体存活和视力所必需的。 具体而言，JNCL HIPSC-RPE显示微绒毛的混乱/气囊和POS的吞噬作用受损 与对照HIPSC-RPE细胞相比。值得注意的是，与RPE微绒毛功能障碍一致和POS降低 在JNCL HIPSC-RPE细胞中的摄取，JNCL患者眼中的RPE减少了脂肪霉素（分解 POS消化产物）。总之，这些数据表明“ Cln3是RPE微绒毛居民蛋白 调节JNCL中的POS摄取和CLN3功能障碍会导致POS吞噬作用受损，然后 降低了JNCL RPE细胞中脂肪霉素的积累。”为了检验该假设，在这个项目中，我们建议 对照和JNCL HIPSC-RPE细胞的员工超微结构和分子研究。具体来说，我们将首先 利用免疫组织化学，rNascope和亚细胞分级技术来确认 RPE细胞中CLN3的比例与原代（小鼠，猪，人）和对照hipsc- RPE。接下来，我们将比较i）微绒毛结构和分子组成以及ii）POS结合和 通过对照与JNCL HIPSC-RPE细胞进行内部化。此外，将减少的POS吞噬作用相关联 通过JNCL HIPSC-RPE细胞减少脂肪志Cin（自动荧光积累），在体内JNCL RPE中， 我们将评估平行培养物中自动荧光积累（细胞表面与细胞内）的模式 慢性POS喂养后的对照和JNCL HIPSC-RPE（1-3个月）。我们还将验证我们的发现 使用JNCL小鼠和猪模型的初级RPE/POS患者HIPSC-RPE细胞。最后，我们将使用 在纵向研究中，JNCL患者的体内视网膜成像记录了存在和数量 在光感受器RPE细胞层中与光感受器与RPE细胞的关系中的自动荧光/脂肪霉素 患者通过该疾病的损失。总之，这项研究所获得的知识将提供 i）CLN3在RPE生理学和ii）RPE功能障碍的合理贡献的新见解 致JNCL视网膜病理生理学。 呢